Objective: Heterozygous mutations in the GBA1 gene elevate the risk of Parkinson disease and dementia with Lewy bodies; both disorders are characterized by misprocessing of a-synuclein (SNCA). A loss in lysosomal acid-bglucosidase enzyme (GCase) activity due to biallelic GBA1 mutations underlies Gaucher disease. We explored mechanisms for the gene's association with increased synucleinopathy risk. Methods: We analyzed the effects of wild-type (WT) and several GBA mutants on SNCA in cellular and in vivo models using biochemical and immunohistochemical protocols. Results: We observed that overexpression of all GBA mutants examined (N370S, L444P, D409H, D409V, E235A, and E340A) significantly raised human SNCA levels to 121 to 248% of vector control (p < 0.029) in neural MES23.5 and PC12 cells, but without altering GCase activity. Overexpression of WT GBA in neural and HEK293-SNCA cells increased GCase activity, as expected (ie, to 167% in MES-SNCA, 128% in PC12-SNCA, and 233% in HEK293-SNCA; p < 0.002), but had mixed effects on SNCA. Nevertheless, in HEK293-SNCA cells high GCase activity was associated with SNCA reduction by 32% (p ¼ 0.009). Inhibition of cellular GCase activity (to 8-20% of WT; p < 0.0017) did not detectably alter SNCA levels. Mutant GBA-induced SNCA accumulation could be pharmacologically reversed in D409V-expressing PC12-SNCA cells by rapamycin, an autophagy-inducer ( 40%; 10lM; p < 0.02). Isofagomine, a GBA chaperone, showed a related trend. In mice expressing two D409Vgba knockin alleles without signs of Gaucher disease (residual GCase activity, !20%), we recorded an age-dependent rise of endogenous Snca in hippocampal membranes (125% vs WT at 52 weeks; p ¼ 0.019). In young Gaucher disease mice (V394Lgbaþ/þ//prosaposin[ps]-null//ps-transgene), which demonstrate neurological dysfunction after age 10 weeks (GCase activity, 10%), we recorded no significant change in endogenous Snca levels at 12 weeks of age. However, enhanced neuronal ubiquitin signals and axonal spheroid formation were already present. The latter changes were similar to those seen in three week-old cathepsin D-deficient mice.Interpretation: Our results demonstrate that GBA mutants promote SNCA accumulation in a dose-and timedependent manner, thereby identifying a biochemical link between GBA1 mutation carrier status and increased synucleinopathy risk. In cell culture models, this gain of toxic function effect can be mitigated by rapamycin. Loss in GCase activity did not immediately raise SNCA concentrations, but first led to neuronal ubiquitinopathy and axonal spheroids, a phenotype shared with other lysosomal storage disorders. ANN NEUROL 2011;69:940-953 View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.22400 Additional Supporting Information can be found in the online version of this article. 940

Preadipocyte differentiation in culture is driven by an insulin and cAMP dependant transcriptional cascade which induces the bzip transcription factors C/EBPbeta and C/EBPdelta. We have previously shown that glucocorticoid treatment, which strongly potentiates this differentiation pathway, stimulates the titration of the corepressor histone deacetylase 1 (HDAC1) from C/EBPbeta. This results in a dramatic enhancement of C/EBPbeta-dependent transcription from the C/EBPalpha promoter, concomitant with potentiation of preadipocyte differentiation. Here, we show that C/EBPbeta is acetylated by GCN5 and PCAF within a cluster of lysine residues between amino acids 98-102 and that this acetylation is strongly induced by glucocorticoid treatment. Arginine substitution of the lysine residues within the acetylation motif of C/EBPbeta prevented acetylation and blocked the ability of glucocorticoids to enhance C/EBPbeta-directed transcription and to potentiate C/EBPbeta-dependent preadipocyte differentiation. Moreover, acetylation of C/EBPbeta appeared to directly interfere with the interaction of HDAC1 with C/EBPbeta, suggesting that PCAF/GCN5-dependent acetylation of C/EBPbeta serves as an important molecular switch in determining the transcriptional regulatory potential of this transcription factor.

Methods: Using heterozygous and homozygous GBA1 D409V knockin mouse astrocytes, we conducted rigorous biochemical and image-based analyses of lysosomal function and morphology. We also examined basal and evoked cytokine response at the transcriptional and secretory levels.Results: The D409V knockin astrocytes manifested broad deficits in lysosomal morphology and function, as expected. This, however, is the first study to show dramatic defects in basal and TLR4-dependent cytokine production. Albeit to different extents, both the lysosomal dysfunction and inflammatory responses were normalized by inhibition of LRRK2 kinase activity, suggesting functional intracellular crosstalk between glucocerebrosidase and LRRK2 activities in astrocytes.Conclusions: These data demonstrate novel pathologic effects of a GBA1 mutation on inflammatory responses in astrocytes, indicating the likelihood of broader immunologic changes in GBA-PD patients. Our findings support the involvement of non-cell-autonomous mechanisms contributing to the pathogenesis of GBA1-linked PD and identify new opportunities to correct these changes with pharmacological intervention.

With the advent of the genetic era in Parkinson’s disease (PD) research in 1997, α-synuclein was identified as an important player in a complex neurodegenerative disease that affects >10 million people worldwide. PD has been estimated to have an economic impact of $51.9 billion in the US alone. Since the initial association with PD, hundreds of researchers have contributed to elucidating the functions of α-synuclein in normal and pathological states, and these remain critical areas for continued research. With this position paper the authors strive to achieve two goals: first, to succinctly summarize the critical features that define α-synuclein’s varied roles, as they are known today; and second, to identify the most pressing knowledge gaps and delineate a multipronged strategy for future research with the goal of enabling therapies to stop or slow disease progression in PD.

Glucocorticoids provide an adipogenic stimulus that is most obvious in the truncal obesity of patients with Cushing's syndrome. Glucocorticoid treatment also strongly potentiates the differentiation of human preadipocytes in culture. However, the molecular basis of these stimulatory effects remains to be defined. In this study, we provide a detailed analysis of the specific contribution of glucocorticoid treatment to the differentiation of primary human preadipocytes cultured in chemically defined medium. Contrary to previous descriptions of glucocorticoids being required throughout the course of differentiation, our results show that glucocorticoid treatment is stimulatory only during the first 48 h of differentiation. Furthermore, stimulation by glucocorticoids and the peroxisome proliferator activator receptor-gamma agonist troglitazone is mediated sequentially. Several details of the early events in the differentiation of human preadipocytes and the contribution of steroid to these events differ from the responses observed previously in murine preadipocyte models. First, glucocorticoid treatment stimulated the early accumulation of CCAAT enhancer binding protein-beta (C/EBPbeta) in primary human preadipocytes. Second, induction of C/EBPalpha in primary human preadipocytes was noted within 4 h of adipogenic stimulus, whereas C/EBPalpha induction is not detected until 24-48 h in the murine 3T3 L1 preadipocyte model. Remarkably, by contrast to human primary preadipocytes, which do not undergo postconfluent mitosis, 3T3 L1 murine preadipocytes stimulated to differentiate under chemically defined conditions required glucocorticoids to survive the clonal expansion that precedes terminal differentiation, revealing a novel signal imparted by glucocorticoids in this immortalized murine cell system.

Variants in the leucine-rich repeat kinase-2 (LRRK2) gene are associated with Parkinson’s disease, leprosy, and Crohn’s disease, three disorders with inflammation as an important component. Because of its high expression in granulocytes and CD68-positive cells, LRRK2 may have a function in innate immunity. We tested this hypothesis in two ways. First, adult mice were intravenously inoculated with Salmonella typhimurium, resulting in sepsis. Second, newborn mouse pups were intranasally infected with reovirus (serotype 3 Dearing), which induced encephalitis. In both mouse models, wild-type Lrrk2 expression was protective and showed a sex effect, with female Lrrk2-deficient animals not controlling infection as well as males. Mice expressing Lrrk2 carrying the Parkinson’s disease–linked p.G2019S mutation controlled infection better, with reduced bacterial growth and longer animal survival during sepsis. This gain-of-function effect conferred by the p.G2019S mutation was mediated by myeloid cells and was abolished in animals expressing a kinase-dead Lrrk2 variant, p.D1994S. Mouse pups with reovirus-induced encephalitis that expressed the p.G2019S Lrrk2 mutation showed increased mortality despite lower viral titers. The p.G2019S mutant Lrrk2 augmented immune cell chemotaxis and generated more reactive oxygen species during virulent infection. Reovirus-infected brains from mice expressing the p.G2019S mutant Lrrk2 contained higher concentrations of α-synuclein. Animals expressing one or two p.D1994S Lrrk2 alleles showed lower mortality from reovirus-induced encephalitis. Thus, Lrrk2 alleles may alter the course of microbial infections by modulating inflammation, and this may be dependent on the sex and genotype of the host as well as the type of pathogen.

Braak and Del Tredici have proposed that typical Parkinson disease (PD) has its origins in the olfactory bulb and gastrointestinal tract. However, the role of the olfactory system has insufficiently been explored in the pathogeneses of PD and Alzheimer disease (AD) in laboratory models. Here, we demonstrate applications of a new method to process mouse heads for microscopy by sectioning, mounting, and staining whole skulls (‘holocranohistochemistry’). This technique permits the visualization of the olfactory system from the nasal cavity to mitral cells and dopamine-producing interneurons of glomeruli in the olfactory bulb. We applied this method to two specific goals: first, to visualize PD- and AD-linked gene expression in the olfactory system, where we detected abundant, endogenous α-synuclein and tau expression in the olfactory epithelium. Furthermore, we observed amyloid-β plaques and proteinase-K-resistant α-synuclein species, respectively, in cranial nerve-I of APP- and human SNCA-over-expressing mice. The second application of the technique was to the modeling of gene–environment interactions in the nasal cavity of mice. We tracked the infection of a neurotropic respiratory-enteric-orphan virus from the nose pad into cranial nerves-I (and -V) and monitored the ensuing brain infection. Given its abundance in the olfactory epithelia, we questioned whether α-synuclein played a role in innate host defenses to modify the outcome of infections. Indeed, Snca-null mice were more likely to succumb to viral encephalitis versus their wild-type littermates. Moreover, using a bacterial sepsis model, Snca-null mice were less able to control infection after intravenous inoculation with Salmonella typhimurium. Together, holocranohistochemistry enabled new discoveries related to α-synuclein expression and its function in mice. Future studies will address: the role of Mapt and mutant SNCA alleles in infection paradigms; the contribution of xenobiotics in the initiation of idiopathic PD; and the safety to the host when systemically targeting α-synuclein by immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00702-017-1726-7) contains supplementary material, which is available to authorized users.

Fifty-five years after the concept of dopamine replacement therapy was introduced, Parkinson disease (PD) remains an incurable neurological disorder. To date, no disease-modifying therapeutic has been approved. The inability to predict PD incidence risk in healthy adults is seen as a limitation in drug development, because by the time of clinical diagnosis ≥ 60% of dopamine neurons have been lost. We have designed an incidence prediction model founded on the concept that the pathogenesis of PD is similar to that of many disorders observed in ageing humans, i.e. a complex, multifactorial disease. Our model considers five factors to determine cumulative incidence rates for PD in healthy adults: (i) DNA variants that alter susceptibility (D), e.g. carrying a LRRK2 or GBA risk allele; (ii) Exposure history to select environmental factors including xenobiotics (E); (iii) Gene-environment interactions that initiate pathological tissue responses (I), e.g. a rise in ROS levels, misprocessing of amyloidogenic proteins (foremost, a-synuclein) and dysregulated inflammation; (iv) sex (or gender; G); and importantly, (v) time (T) encompassing ageing-related changes, latency of illness and propagation of disease. We propose that cumulative incidence rates for PD (P R ) can be calculated in healthy adults, using the formula: P R (%) = (E + D + I) 9 G 9 T. Here, we demonstrate six case scenarios leading to young-onset parkinsonism (n = 3) and late-onset PD (n = 3). Further development and validation of this prediction model and its scoring system promise to improve subject recruitment in future intervention trials. Such efforts will be aimed at disease prevention through targeted selection of healthy individuals with a higher prediction score for developing PD in the future and at disease modification in subjects that already manifest prodromal signs.